Technical Abstract:
The goal of our study is to optimize n-butanol production from n-butyrate, using pure cultures of solventogenic Clostridia. In addition to butyrate as a substrate, glucose was used as a source of energy and to provide reducing equivalents to facilitate the conversion. To prevent product inhibition and to ensure continuous fermentation in chemostats, butanol was removed and concentrated by gas stripping and condensing. First, we performed a strain comparison of 10 Clostridia strains that had been reported to be satisfactory solvent producers and found that Clostridium saccharoperbutylacetonicum N1-4 (ATCC 27021) was able to generate the highest concentration of butanol with glucose as the substrate. Next, we determined the optimum conditions for maintenance and growth for strain N1-4. A pH of 4.8 was determined as the optimum pH to run the culture with constant solventogenesis (i.e., acid to solvent conversion) and sufficient growth rate, viability, and cell yield. It is already known that triggering solventogenesis is dependent on the culture pH and the total organic-acid concentration (i.e., butyrate) because the concentration of undissociated acids must be high enough. For example, with strain N1-4 we determined that butyric acid triggers solventogenesis at a total butyrate concentration of 2 g/L at a pH of 4.8, while this was 10 g/L at a pH of 6. The concentration of undissociated butyric acid to maintain N1-4 in the solventogenic state is not equal at these different pHs with a concentration of ~0.8 g/L at a pH of ~5 and a concentration <0.5 g/L at relatively low and high pH values. We were able to trigger solventogenesis at pH values exceeding six when we maintained high enough concentrations of butyrate. All of these studies were performed in batch cultures to find optimum conditions for continuous fermentation. Second, we operated several continuous cultures with gas stripping at a pH of 4.8, but none of the cultures remained stable for longer than 4 days because of oscillations between solventogenesis and acidogenesis (high and low butyrate concentration in the reactor, respectively). We solved this problem with a two-stage chemostat setup with butyrate fed to the second stage in pH-auxostat mode to maintain a stable pH and butyrate concentration. Under these conditions, we were able to maintain the culture in a constant solventogenic mode for more than 2 weeks while steadily converting butyrate into butanol. We are now further optimizing this continuous fermentation process to reduce the amount of sugars that are necessary for energy and reducing power.